Molecular genetic analysis of bilateral convergent strabismus with exophthalmus in German Brown cattle

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Molecular genetic analysis of bilateral convergent strabismus with

exophthalmus in German Brown cattle

INAUGURAL-DISSERTATION zur Erlangung des Grades eines

Doktors der Veterinärmedizin - Doctor medicinae veterinariae -

( Dr. med. vet. )

vorgelegt von Steffen Martin Fink

Mainz Hannover 2009

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Scientific supervisor: Univ.-Prof. Dr. Ottmar Distl

Institut für Tierzucht und Vererbungsforschung

Examiner: Univ.-Prof. Dr. Ottmar Distl Co-examiner: Univ. Prof. Dr. Hans-J. Hedrich Oral examination: 18.05.2009

This work was supported by grands from the German Research Council, DFG, Bonn, Germany (DI 333/7-3).

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Dedicated to my family and

especially to my deceased grandma

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Parts of this work have been published or submitted for publication in the following journals:

1. Animal Genetics

2. Molecular Vision

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List of abbreviations

A Adenine Acc. No. accession number

APS ammonium persulphate

AT annealing temperature

BAC bacterial artificial chromosome

BCSE Bilateral convergent strabismus with exophthalmus BLAST basic local alignment search tool

BLASTN basic local alignment search tool nucleotide bp basepairs

BTA Bos taurus autosome C cytosine

C10orf2 chromosome 10 open reading frame 2 cDNA complementary deoxyribonucleic acid

CDS coding sequence

cen centromer

CFEOM congenital fibrosis of the extraocular muscles cM centiMorgan

CPT1C carnitine palmityltransferase 1C cR centiRay

DFG Deutsche Forschungsgemeinschaft (German Research Council) DHDH dihydrodiol dehydrogenase dimeric

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

dNTPs deoxy nucleoside 5’ triphosphates (N is A, C, G, or T) DRS/DURS duane retraction syndrome

EBI European Bioinformatics Institute EDTA ethylenediamine tetraacetic acid

EMBL European Molecular Biology Labaratory EST expressed sequence tag

F forward

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G guanine GLM general linear model gss genomic survey sequence HET heterozygosity

Ho observed heterogosity

HSA Homo sapiens autosome

HWE Hardy-Weinberg equilibrium IBD identical by descent

Indel insertion/deletion

IRD infrared dye

kb kilobase

KIF21A kinesin family member 21A

LD linkage disequilibrium

LOD logarithm of the odds M molar M. musculus

MARC meat animal research center Mb megabase

MERLIN multipoint engine for rapid likelihood inference mRNA messenger ribonucleic acid

MS microsatellite NaCl Sodiumchloride

NAH2PO4 natriumdihydrogenphosphat NA2HPO4 dinatriumhydrogenphosphat

NALP7 NLR family, pyrin domain containing 7

NCBI National Center for Biotechnology Information no. Number

NPL nonparametric linkage ORF open reading frame

p error probability

PCR polymerase chain reaction

PEO progressive external ophthalmoplegia

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PIC polymorphism information content POLG polymerase (DNA directed), gamma POS position

QTL quantitative trait locus R reverse

RDH13 retinol dehydrogenase 13

RH radiation hybrid

RNA ribonucleic acid

rpm rounds per minute

SAS statistical analysis system SDS sodium dodecyl sulfate

SLC25A4 solute carrier family 25, member 4 SNP single nucleotide polymorphism STS sequence-tagget site SYT synaptogamin

T thymine

Ta annealing temperature

TBE tris-borate-ethylenediamine tetraacetic acid TCF3 transcription factor 3

TE tris-ethylenediamine tetraacetic acid TEMED N,N,N’,N’-tetramethylenediamine Tel telomer

TFPT TCF3 fusion partner TNNT1 troponin T type 1

USDA united state department of agriculture UTR untranslated region

UV ultraviolet w/v weight to volume

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CHAPTER 1

Introduction

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1 Introduction

Bilateral convergent strabismus with exophthalmus (BCSE) is a heritable eye disorder affecting many cattle breeds worldwide. German Brown cattle show an incidence of 0.9% for BCSE. The defect is characterised by a symmetric anterior- medial rotation of both eyeballs associated with a slight to severe laterodorsal exophthalmus. The pupils disappear in the nasal corner of the orbits and the amount of the visible parts of sclera in the temporal angle of the orbit increases, which finally ends in complete blindness. Due to the progression of BCSE, the affected animals show more and more changes in the behaviour like shying, panic, aggressiveness and reluctance to walk. Thus these animals are difficult to handle in everyday situation. Due to the severly limited use of an organ, breeding with animals which are known or suspected to be carrier of BCSE is not allowed by paragraph 11b of the German animal welfare laws. Due to the fact that most animals develop BCSE in the adulthood, often after first breeding, it is impossible to eliminate BCSE in the population alone by exclusion of affected animals from breeding. An autosomal dominant major gene influenced by different genetic factors is the most probable mode of inheritance. In case of an affected sire which is used for artificial insemination the defect can spread among the population very fast. Consequently, a molecular genetic diagnosis of carriers is urgently needed.

The objectives of the present study were to identify the genomic regions harbouring the gene loci responsible for BCSE by linkage analysis and further fine mapping of the detected BCSE regions with newly developed and published single nucleotide polymorphisms (SNPs). Therefore we performed single marker association tests and estimation of haplotype association to refine the BCSE regions and to detect potential candidate genes for BCSE.

For some genes which were chosen due to their location within the linked BCSE interval, expression profile and known function in other animals or human, cDNA and mutation analyses were carried out. We sequenced the coding region of these genes in affected and unaffected German Brown cattle and searched these sequences for polymorphisms, which were tested for association with BCSE.

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Overview of chapter contents

Chapter 2 contains the whole genom scan performed on 10 families and the linkage analysis to determine the genomic regions responsible for BCSE in German Brown cattle

In Chapter 3 and 4 the results of the association of QTL regions on BTA5 and BTA18 are shown. Further fine mapping of both BCSE loci are carried out by haplotype association analysis. In addition, the molecular characterization and mutation analysis of nine candidate genes for BCSE is described.

Chapter 5 provides a general discussion and conclusions referring to Chapters 1-4.

Chapter 6 is a concise English summary of this thesis, while Chapter 7 is an expanded, detailed German summary which takes into consideration the overall research context.

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CHAPTER 2

Linkage of bilateral convergent strabismus with exophthalmus (BCSE) to BTA5 and BTA18 in German Brown cattle

S. Mömke, S. Fink, A. Wöhlke, C. Drögemüller and O. Distl

Institute for Animal Breeding and Genetics, Universtiy of Veterinary Medicine Hannover, Foundation, Germany

Published in: Animal Genetics 39 (2008) 544-549.

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2 Linkage of bilateral convergent strabismus with exophthalmus (BCSE) to BTA5 and BTA18 in German Brown cattle

2.1 Summary

Bilateral convergent strabismus with exophthalmus (BCSE) is a widespread inherited eye defect in several cattle populations. Its progressive condition often leads to blindness in affected cattle and decreases their usability. Furthermore, the German animal welfare laws prevent breeding with animals whose progeny are expected to be affected by genetic defects. Identifying genes involved in the heredity of BCSE should lead to insights in the molecular pathogenesis of this eye disease and permit to establish a genetic test for this disease. A whole genome scan for 10 families containing a total of 159 genotyped individuals identified two BCSE loci. One BCSE locus mapped to the centromeric region on bovine chromosome (BTA) 5 and the other BCSE locus to the telomeric region of BTA18. Thus, it is possible, that two genes are involved in the development of BCSE. Also, one of these loci could be causal for the development of BCSE and the other locus could affect the progression and severity of the defect.

Keywords cattle, microsatellite. ocular disorder, quantitative trait loci, strabismus

2.2 Introduction

Bilateral convergent strabismus with exophthalmus (BCSE) occurs in many cattle breeds including Holsteins, German Brown and Fleckvieh cattle (Distl 1993; Distl &

Gerst 2000; Gerst & Distl 1997, 1998; Mömke & Distl 2007). BCSE is characterised by a bilateral symmetric anterior-medial rotation of the eyes associated with a slight to severe protrusion of the eyeballs. Both eyes are fixed in this position and the animals are not able to move their eyeballs. The condition is progressive and in advanced stages leads to blindness caused by rotation of the pupil into the orbit. The defect can cause changes in the behaviour of the affected animals such as

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aggressiveness, shying and panic in everyday situations. This eye anomaly is incurable (Mömke & Distl 2007). Neither associations between BCSE and milk performance traits nor associations of BCSE with breeding values for milk production traits have been evident (Distl & Gerst 2000).

A complex segregation analysis gave evidence for a single autosomal dominant major gene responsible for the phenotypic expression of BCSE in German Brown cattle (Distl 1993). Progressive external ophthalmoplegia (PEO) in man refers to a group of disorders characterised by ptosis and slowly progressive bilateral immobility of the eyes (Sorkin et al. 1997) and shows striking similarities to BCSE in cattle.

Therefore, the three genes POLG, SLC25A4, C10orf2 responsible for PEO were chosen as candidate genes for BCSE in cattle. However, all these genes could be ruled out for bovine BCSE (Hauke 2003; Mömke & Distl 2007).

Breeding with animals, which are known or suspected to be carriers of BCSE is forbidden by animal welfare laws in Germany, due to the severely limited use of the eyes in affected individuals. The onset of the defect can sometimes be late in life and often first signs of the defect are not noticed prior to first breeding. Thus, prevention of BCSE cannot be achieved alone by exclusion of affected animals from breeding.

Consequently, a molecular genetic diagnosis of carriers is urgently needed. We mapped the genomic regions on bovine chromosomes 5 and 18 harbouring gene loci responsible for BCSE.

2.3 Material and methods

2.3.1 Sampling of animals and pedigree structure

For the linkage analysis, we included blood, semen or hair samples from 159 German Brown cattle belonging to two large paternal half-sib and eight smaller families segregating for BCSE (Table S1). The first half-sib family (family 1) consisted of the affected sire, 14 affected daughters, 15 unaffected daughters and twelve dams, of which three were affected. The second half-sib family (family 2) included the

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affected sire and 26 affected and 19 unaffected daughters. The remaining eight families consisted of two to eleven affected, related females and randomly selected unaffected relatives. Because of the late onset of the disease, we primarily used affected individuals in our analysis. The average family size was 30.1 individuals. In the average, the families included 3.9 generations, ranging from 2 to 8. The prevalence of BCSE was 56.6 % in the genotyped animals. The average age at which BCSE was first detected was 6.1 years for the families 3 to 10, 6.9 years for family 1 and 3.7 years for family 2.

Genomic DNA from EDTA blood samples was extracted using the QIAamp 96 Spin Blood kit (Qiagen, Hilden, Germany). For hair specimens, the DNeasy Tissue kit (50) (Qiagen) was used and for semen samples, the Nucleon BACC2-Kit for blood and cell cultures (AmershamBiosciences, Freiburg, Germany) was applied.

2.3.2 Microsatellite markers

For a whole genome scan we selected 164 polymorphic microsatellite markers to achieve a uniform coverage of all bovine autosomes comprising 2808.9 cM with an average pair-wise distance of 19.9 cM (Ihara et al. 2004). We used 5.7 markers per chromosome in the average. In the second step of the genome scan, six genomic regions located on bovine chromosomes 5, 6, 8, 16, 18 and 22 with putative linkage and highest Zmeans and LOD scores were scanned with a total of 43 additional microsatellite markers. In the average, the respective six chromosomes were covered by 13.2 markers, each with a mean pair-wise distance of 7.7 cM. The same families were used as in the whole genome scan.

2.3.3 PCR

All PCR reactions were carried out in 12 µl reaction mixtures containing 2 µl genomic DNA (10 ng/µl), 1.2 µl 10x PCR buffer, 0.24 µl DMSO, 0.5 µl of each primer (10 pmol/µl), 0.2 µl dNTPs (5 mM each) and 0.1 µl Taq Polymerase (5 U/µl) (Qbiogene, Heidelberg, Germany). To increase efficiency, 102 primer pairs were pooled into PCR multiplex groups of two to five markers, and the 62 remaining primer pairs were amplified separately. One primer of each pair was endlabeled with fluorescent

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IRD700 or IRD800. For amplification, PTC 100™ or PTC 200™ thermal cyclers (MJ Research, Watertown, MA, USA) and a general PCR program with variable annealing temperature (AT) were used. The reaction started with denaturing all samples at 94°C for 4 min followed by 36 cycles comprising denaturation for 30 s at 94°C, annealing for 45 s at AT (52-70°C) and extension for 45 s at 72°C. The PCR was completed with a final cooling at 4°C for 10 min. For the analysis of the marker genotypes, the PCR products were size-fractionated by gel electrophoresis on automated sequencers (LI-COR 4200 and 4300, Lincoln, NE, USA) using 6%

polyacrylamide denaturing gels (RotiphoreseGel 40, Roth, Karlsruhe, Germany).

2.3.4 Development of new markers for fine mapping

A search for SNPs (Table S2) was conducted using the sires of the half sib families 1 and 2. Genomic sequences of the BAC clones CH240-433A8, CH240-34B7 and RP42-155H10, which gave significant hits to the syntenic region of HSA12 and HSA19, respectively, were used, as well as bovine gene sequences (Larkin et al.

2003) or BAC end sequences which were assigned to the bovine genome region of interest on BTA5 and BTA18 RH3,000 comparative maps (Mömke et al. 2005a, 2005b). After masking the repetitive sequences, primers were designed using the Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The amplicons were sequenced on a MegaBACE 1000 automated sequencer (GE Healthcare, Freiburg, Germany). The sequencing reaction was carried out using the DYEnamic ET Terminator Cycle Sequencing kit (GE Healthcare) for both sires of the two half-sib families. If a heterozygous SNP was found for one or both sires, all progeny and dams of the respective families were genotyped for that SNP. For fine mapping, in addition to the microsatellite markers used for the whole genome analysis, these SNPs were incorporated into the linkage analysis according to the RH mapping data (Mömke et al. 2005a, 2005b). In total, we identified 13 SNPs, which were heterozygous for one or both of the two sires that founded the half-sib families in our material.

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2.3.5 Statistical analysis

To detect linkage, non-parametric multipoint linkage analysis was employed (Kong &

Cox 1997; Kruglyak et al. 1996; Whittemore & Halpern 1994). The Whittemore and Halpern NPL pairs statistics Zmean and LOD score were used to test for linkage using allele sharing among affected pedigree members. Non-parametric linkage analyses do not require assumptions on the mode of inheritance and the genetic parameters of the specified model and so this approach should be useful for traits under unknown inheritance models. We employed multipoint analyses in order to use marker information from the whole chromosome through linked informative markers and to increase power of linkage analysis. We determined empirical chromosome- wide significance levels using simulated marker genotypes under the null hypothesis of no linkage to observed phenotypes. The empirical distribution for the 5% error probability was obtained after 10,000 replicates. A significant chromosome-wide co- segregation of marker alleles among affected family members with BCSE was assumed for the empirically determined significance levels below 5%. For the genome-wide type-I error probability (Pgenome-wide), a Bonferroni correction was applied for the chromosome-wide error probability with Pgenome-wide = 1- (1 – Pchromosome-wide)^1/r, where r is the ratio of the length of the respective chromosome harbouring the QTL to the total length of the bovine genome with 2809 cM. We used Bonferroni’s procedure in order to strictly control the overall type-I error rate of genome-wide error probabilities.

The statistical analyses were performed using the MERLIN Software, version 1.0.1 (Abecasis et al. 2002).

2.4 Results

2.4.1 Characteristics of the marker set

The 164 markers used for the whole genome scan had a mean number of 5.9 alleles in our material. The average polymorphism information content (PIC) of this set was

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56.0% and the mean observed heterozygosity 60.0%, so our marker set was appropriate for linkage studies. The PIC was higher than 50% in 110 markers (67.1%). Only ten markers (6.1%) showed a PIC < 25%. The highest average number of alleles was 8.3 for BTA23 and the lowest one was 5.0 for BTA7 and BTA24.

2.4.2 Whole genome scan and further refinement of putative genomic regions

We detected genomic regions on bovine chromosomes 5 and 18 significantly linked with the BCSE phenotype at chromosome-wide error probabilities for Zmeans and LOD scores below 0.05. The most likely position for the locus on BTA18 was mapped distally of microsatellite BMS2785. The BCSE region on BTA5 extended between the markers BP1 at 17.29 cM and BL37 at 52.09 cM. The maximum Zmean and maximum LOD score were at 41.69 cM (BMC1009). On BTA18, the 95% confidence interval spanned the region from 72.01 cM to 84.38 cM with Zmean and LOD score highest at 84.09 cM (TGLA227).

2.4.3 Fine mapping

Thirteen SNPs (Table S2) as well as a total of six additional microsatellite markers were integrated into the bovine maps of BTA5 and BTA18 and the data were re- analyzed for linkage with BCSE. Including these markers into the linkage analysis did not affect the position of the BCSE regions identified in the whole genome scan but increased the Zmeans and LOD scores and decreased the 95% confidence intervals.

On BTA5, the marker AGLA293 showed the highest Zmean (4.18) with a chromosome-wide error probability of P < 0.001 and a genome-wide error probability of P < 0.001 at 32.25 cM. The 95% confidence interval now spanned from 17.29 cM (BP1) to 47.0 cM (VDR_SNP). On BTA18, the 95% confidence interval spanned from 72.01 cM (BMS2785) to 78.84 cM (BM6507). The marker DIK5109 peaked with a Zmean of 2.82 (chromosome-wide P = 0.002, genome-wide P = 0.06) (Tables 1 and 2). This marker was located at 77.6 cM. After this conjoined analysis, the two half-sib families were scanned separately. For family 1, the 95% confidence interval on BTA5

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ranged from 20.11 cM (DIK2828) to 47.00 cM (VDR_SNP), and on BTA18 it extended from 55.53 cM (BMS2639) to the end of the chromosome (DIK4013 at 84.38 cM). The highest Zmeans on BTA5 were found at 32.25 cM (AGLA293) with a maximum Zmean of 4.51 (chromosome-wide and genome-wide P < 0.001) and for BTA18 the marker DIK5109 reached a Zmean of 6.08 (chromosome-wide and genome-wide P < 0.001) at 77.6 cM. Regarding family 2, the 95% confidence interval for BTA5 extended between the SYT1_SNP (8.0 cM) and OARFCB5 (35.28 cM). The Zmeans peaked with values of 1.88 (chromosome-wide P = 0.03) at 30.04 cM (DCN_SNP) and 30.13 cM (BL23) (Tables 1 and 2). No significant linkage was evident on BTA18 for family 2, but it has to be mentioned, that almost all markers on the telomeric region of this chromosome were homozygous for the common sire of this family.

We applied the best option of MERLIN software to reconstruct the most likely haplotypes in the progeny of the two sires. In paternal half-sib family 1, twelve of the 14 affected siblings received the same BCSE-linked haplotype on BTA5 from their sire. In paternal half-sib family 2, 20 of the 26 affected siblings showed the same BCSE-linked haplotype on BTA5. Haplotype analysis of BTA18 revealed that 13 of the 14 affected siblings of sire 1 got the same paternal BCSE-linked haplotype. Only one animal showed the alternative haplotype. In total, eleven affected daughters in paternal half-sib family 1 inherited the haplotypes linked with BCSE on both chromosomes and the remaining three daughters inherited the susceptible haplotype on one of both chromosomes.

2.5 Discussion

The existence of two putative loci involved in the development of BCSE in German Brown cattle was shown by linkage analysis. These putative BCSE loci were located on the bovine chromosomes 5 and 18, respectively. The two loci were mapped on BTA5 between the markers BP1 (17.29 cM) and VDR_SNP (47.0 cM), and on the telomeric end of BTA18 between the markers BMS2785 (72.01 cM) and BM6507 (78.84 cM). Haplotype analysis allowed us further corroboration of these two linked

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BCSE regions. On BTA5, the minimum BCSE-linked haplotype extended to an interval of 11.56 cM between 30.13 and 41.69 cM. On BTA18, the minimum BCSE- linked region extended between 72.01 and 78.84 cM spanning 6.83 cM.

We developed new SNP markers for further refinement of the two genomic regions linked to the BCSE phenotype. The SNP markers turned out to be mostly unambiguous and useful. The inclusion of the SNP markers led to higher peaks of the test statistic Zmean, lower error probabilities and smaller confidence intervals.

Distl (1993) proved a single autosomal dominant major gene responsible for the phenotypic expression of BCSE, so it is possible that only one of the two chromosomes carries the gene causing BCSE. The second gene could influence the severity of BCSE and the age of onset. A possible indication for this hypothesis could be found in the differences in age of onset between the two paternal half-sib families (families 1 and 2). While most of the animals of family 2 showed an early onset of the eye defect with about three to four years, most of the progeny in family 1 developed signs of BCSE not prior to an age of six years. The heterogeneity of the Zmeans for BTA18 between these two families supports this. So the BCSE locus on BTA18 may accelerate the progression of BCSE. In family 2, no significant linkage for BCSE could be calculated for BTA18. This could be due to a homozygous status of the mutation in the common sire. On the other hand, the sire of family 2 could lack a mutation on BTA18, which retards the onset and progression of BCSE.

When regarding all pedigrees, most affected individuals carry marker alleles that are linked to BCSE on both chromosomal BCSE regions. However, three out of the 14 affected animals in the paternal half-sib family 1 showed the defect, even if they got only one of the paternal haplotypes linked to BCSE. Therefore, it could also be assumed, that these two genes act independently of each other and that each locus can cause BCSE. At least one copy of a BCSE locus would be sufficient for the development of BCSE.

However, these animals could also have inherited the BCSE causing haplotype from their dams as two of these dams were already culled and thus could not be sampled.

One dam was recorded as negative for BCSE, but the cow could still carry the susceptible genotype due to the sometimes very late onset of the disease.

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Genomic regions linked to BCSE in cattle were found on BTA5 and BTA18. The most likely position for the BCSE region was determined between 17.29 and 47.0 cM on BTA5 and between 72.01 and 78.84 cM on BTA18 using linkage analysis. The next step in this study will be to identify positional candidate genes and intragenic SNPs for an association screen in large samples of cases and controls.

2.6 Acknowledgements

This study was supported by a grant of the German Research Council, DFG, Bonn, Germany (DI 333/7-2). The authors thank all breeders and veterinarians for their readiness to support collection of blood samples of affected animals and controls.

2.7 References

Abecasis G.R., Cherny S.S., Cookson W.O. & Cardon L.R. (2002) Merlin – rapid analysis of dense genetic maps using sparse gene flow trees. Nature Genetics 30, 97-101.

Distl O. (1993) Analysis of pedigrees in dairy cattle segregating for bilateral strabismus with exophthalmus. Journal of Animal Breeding and Genetics 110, 393-400.

Distl O. & Gerst M. (2000) Association analysis between bilateral convergent strabismus with exophthalmus and milk production traits in dairy cattle. Journal of Veterinary Medicine A 47, 31-6.

Gerst M. & Distl O. (1997) Einflüsse auf die Dissemination des bilateralen Strabismus convergens mit Exophthalmus beim Rind. Archiv für Tierzucht 40, 401-12.

Gerst M. & Distl O. (1998) Verbreitung und Genetik des bilateralen Strabismus convergens mit Exophthalmus beim Rind. Tierärztliche Umschau 53, 6-15.

Hauke, G. (2003) Candidate gene analysis for bilateral convergent strabismus with exophthalmus in German Brown cattle. Thesis. University of Veterinary Medicine Hannover.

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Ihara N., Takasuga A., Mizoshita K., Takeda H., Sugimoto M., Mizoguchi Y., Hirano T., Itoh T., Watanabe T., Reed K.M., Snelling W.M., Kappes S.M., Beattie C.W., Bennett G.L. & Sugimoto Y. (2004) A comprehensive genetic map of the cattle genome based on 3802 microsatellites. Genome Research 14, 1987-98.

Kong A. & Cox N.J. (1997) Allele-sharing models: LOD scores and accurate linkage tests. American Journal of Human Genetics 61, 1179-88.

Kruglyak L., Daly M.J., Reeve-Daly M.P. & Lander E.S. (1996) Parametric and nonparametric linkage analysis: a unified multipoint approach. American Journal of Human Genetics 58, 1347-63.

Larkin D.M., Everts-van der Wind A., Rebeiz M., Schweitzer P.A., Bachman S., Green C., Wright C.L., Campos E.J., Benson L.D., Edwards J., Liu L., Osoegawa K., Womack J.E., de Jong P.J. & Lewin H.A. (2003) A cattle human comparative map built with cattle BAC-ends and human genome sequence. Genome Research 13, 1966-72.

Mömke S. & Distl O. (2007) Bilateral convergent strabismus with exophthalmus (BCSE) in cattle: an overview of clinical signs and genetic traits. The Veterinary Journal 173, 272-7.

Mömke S., Drögemüller C., Distl O. (2005a) A high-resolution radiation hybrid map of bovine chromosome 5q1.3-q2.5 compared with human chromosome 12q. Animal Genetics 36, 248-53.

Mömke S., Kuiper H., Spötter A., Drögemüller C. & Distl O. (2005b) A refined radiation hybrid map of the telomeric region of bovine chromosome 18q25-q26 compared with human chromosome 19q13. Animal Genetics 36, 248-53.

Strabismus and mitochondrial defects in chronic progressive external ophthalmoplegia. American Journal of Ophthalmology 123, 235-42.

Whittemore A.S. & Halpern J. (1994) A class of tests for linkage using affected pedigree members. Biometrics 50, 118-27.

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Table 1 Zmeans and their chromosome-wide error probabilities (P) of BTA5 for all families and separately for half-sib families 1 and 2 using non-parametric multipoint linkage analysis for BCSE in German Brown cattle. Chromosome-wide significant Zmeans and their p-values (P) are written in bold.

Position

All families Half-sib family 1 Half-sib family 2

Marker name (cM) Zmean P Zmean P Zmean P

Min* -1.21 0.9 -0.73 0.8 -0.96 0.8 Max* 15.71 <0.001 9.54 <0.001 12.49 <0.001

BMS695 1.17 0.79 0.2 -0.09 0.5 0.85 0.2

BMS6026 6.05 0.89 0.2 -0.23 0.6 1.03 0.2

SYT1_SNP 8.00 0.93 0.2 -0.30 0.6 1.12 0.13 BP1 17.29 1.32 0.09 -0.39 0.7 1.62 0.05

DIK2828 20.11 1.68 0.05 0.14 0.4 1.85 0.03

RM103 29.43 3.02 0.0013 2.38 0.009 1.87 0.03 DCN_SNP 30.04 3.04 0.0012 2.47 0.007 1.88 0.03

BL23 30.13 3.09 0.001 2.56 0.005 1.88 0.03

AGLA293 32.25 4.18 <0.001 4.51 <0.001 1.86 0.03 DIK5002 33.65 4.09 <0.001 4.33 <0.001 1.73 0.04 BM1315 33.66 4.09 <0.001 4.33 <0.001 1.73 0.04 CSAD_SNP 34.00 4.07 <0.001 4.28 <0.001 1.70 0.04 OARFCB5 35.28 3.98 <0.001 4.10 <0.001 1.59 0.06 ILSTS22 38.24 3.76 <0.001 3.82 <0.001 1.35 0.09 BMS321 38.24 3.76 <0.001 3.82 <0.001 1.35 0.09 BMC1009 41.69 3.22 <0.001 3.29 <0.001 1.08 0.14 BMS1898 44.48 2.87 0.002 2.90 0.002 1.05 0.15 VDR_SNP 47.00 1.54 0.06 0.65 0.3 1.12 0.13

BL37 52.09 0.56 0.3 -0.73 0.8 1.01 0.2

KIF21A_SNP 52.10 0.56 0.3 -0.73 0.8 1.01 0.2 BL4 52.40 0.56 0.3 -0.73 0.8 1.02 0.2 BMS1617 56.30 0.59 0.3 -0.73 0.8 1.08 0.14

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Table 1 continued

Position

All families Half-sib family 1 Half-sib family2

Marker name (cM) Zmean P Zmean P Zmean P Min* -1.21 0.9 -0.73 0.8 -0.96 0.8 Max* 15.71 <0.001 9.54 <0.001 12.49 <0.001 HMGA2_SNP 58.00 0.59 0.3 -0.72 0.8 1.09 0.14 LOC644915_SNP 61.00 0.62 0.3 -0.65 0.7 1.08 0.14 BMS1216 78.21 0.47 0.3 -0.11 0.5 0.58 0.3 BM315 103.17 0.48 0.3 0.49 0.3 0.21 0.4 BMS597 125.05 -0.15 0.6 -0.40 0.7 0.09 0.5

*Minimum and maximum achievable values for Zmeans and their P-values in the present linkage analyses. Genome-wide error probabilities below P < 0.001 were obtained for the linked region when a Bonferroni correction (Pgenome-wide = 1- (1 – Pchromosome-wide)^1/r, with r being the length of BTA5 (122 cM) divided by the total bovine genome length) was applied.

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Table 2 Zmeans and their chromosome-wide error probabilities (P) of BTA18 for all families and separately for half-sib family 1 using non-parametric multipoint linkage analysis for BCSE in German Brown cattle. Chromosome-wide significant Zmeans and their p-values (P) are written in bold.

Position All families Half-sib family 1 Marker name (cM) Zmean P Zmean P

Min* -1.20 0.9 -0.73 0.8

Max* 17.34 <0.001 12.37 <0.001

IDVGA31 0.00 -0.18 0.6 -0.24 0.6

BMS2213 24.49 -0.25 0.6 -0.25 0.6

INRA63 47.95 -0.58 0.7 -0.56 0.7

BMS2639 55.53 -0.12 0.5 0.40 0.3

IDVGA55 67.72 0.46 0.3 1.80 0.04

DIK5220 69.82 0.57 0.3 2.08 0.02

KCNJ14_SNP 70.00 0.58 0.3 2.11 0.02

BMS2785 72.01 1.01 0.2 2.75 0.003

DIK2696 76.39 2.37 0.009 5.30 <0.001 BM2078 76.78 2.52 0.006 5.55 <0.001 RDH13_SNP1 77.50 2.79 0.003 6.02 <0.001 RDH13_SNP2 77.51 2.80 0.003 6.03 <0.001 RDH13_SNP3 77.52 2.80 0.003 6.04 <0.001 RDH13_SNP4 77.53 2.80 0.003 6.04 <0.001 DIK5109 77.60 2.82 0.002 6.08 <0.001 PEG_SNP 78.84 2.52 0.006 5.32 <0.001 BM6507 78.84 1.45 0.07 3.53 <0.001 DIK5235 79.47 1.45 0.07 3.37 <0.001

TGLA227 84.09 1.03 0.2 2.30 0.011

DIK4013 84.38 1.03 0.2 2.29 0.011

*Minimum and maximum achievable values for Zmeans and their P-values in the present linkage analyses. Genome-wide error probabilities below P < 0.001 were obtained for the linked region when a Bonferroni correction (P_genome-

wide = 1- (1 – P chromosome-wide)^1/r, with r being the length of BTA18 (84 cM) divided by the total bovine genome length) was applied for half-sib family 1.

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CHAPTER 3

Genes on bovine chromosome 18 associated with bilateral convergent strabismus with exophthalmus in German Brown cattle

S. Fink, S. Mömke, A. Wöhlke and O. Distl

Institute for Animal Breeding and Genetics, Universtiy of Veterinary Medicine Hannover, Foundation, Germany

Published in: Molecular Vision 14 (2008) 1737-1751.

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3

Genes on bovine chromosome 18 associated with bilateral convergent strabismus with exophthalmus in German Brown cattle

3.1 Abstract

Purpose: Bilateral convergent strabismus with exophthalmus (BCSE) is a widespread inherited eye defect in several cattle populations. Its progressive condition often leads to blindness in affected cattle and shortens their length of productive life. Furthermore, breeding with BCSE-affected animals is forbidden by the German animal welfare laws. We performed a mutation and association analysis for three candidate genes (TNNT1, RDH13 and TFPT) which are located within the previously identified BCSE-linked region on the telomeric end of BTA18. In addition, we developed SNPs within these three candidate genes and further nine genes contained in this genomic BCSE-region to perform association analyses with BCSE in German Brown cattle.

Methods: We performed cDNA analyses of all three candidate genes using eye tissues of three affected German Brown cows and three unaffected controls.

Furthermore, we screened the exonic and the adjacent genomic sequences of RDH13, TNNT1 and TFPT using four BCSE-affected and four controls of German Brown cattle. Here, we included all exons of RDH13 and those exons of TNNT1 and TFPT for which SNPs were detected by cDNA analyses. In addition, we developed 21 PCR-products for further 17 genes in the BCSE region and searched them for polymorphisms. All markers detected were genotyped in 48 BCSE-affected German Brown cows and 48 breed and sex matched controls and tested for association with BCSE.

Results: In total, we detected 29 SNPs in twelve genes. In the coding sequence of the three candidate genes we identified ten exonic SNPs and a new splice variant of TNNT1. Four SNPs were associated with the BCSE phenotype in single marker-trait

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analyses. These SNPs were located within the DHDH, CPT1C, TNNT1 and NALP7 genes.

The marker-trait association for haplotypes including five SNPs of the genes CPT1C, SYT5, RDH13 and NALP7 revealed a significant association with BCSE. We identified three individual haplotypes which were significantly associated with BCSE.

These haplotypes spanned the region from 56.05 to 62.87 Mb on BTA18.

Conclusions: The haplotype association analysis corroborated the results of the linkage study that the telomeric end of BTA18 harbors a gene responsible for BCSE and further refines the BCSE region to a 6.82 Mb interval ranging from 56.05 to 62.87 Mb on BTA18.

3.2 Introduction

Bilateral convergent strabismus with exophthalmus (BCSE) is a heritable eye defect which occurs in many cattle breeds, e.g. Jersey, German Fleckvieh, German Holstein and German Brown [1-4]. The incidence of BCSE was estimated to be 0.9% in German Brown cattle [2]. This eye defect is characterized by a progressive, bilateral symmetric anterior-medial rotation of the eyes, associated with a slight to severe protrusion of the eyeballs, which can result in complete blindness. In the development of the bilateral convergent strabismus a defect in the lateral rectus muscle and the retractor bulbi muscle of the eye or in their appendant nerves (Nervus abducens and Nervus oculomotorius) might be involved. The histopathological examination of the nuclei of abducens nerves showed significant differences between BCSE affected and unaffected cows in the number of nerve cells. BCSE affected animals had a decreased number of nerve cells in both nuclear regions (Nuclei n. abducentis dexter and sinister) and this may related with paresis of the M. rectus lateralis and the lateral parts of M. retractor bulbi which is also involved in lateral eye-movement [5]. The histomorphological examination of the lateral and medial rectus muscles of BSCE-affected cows revealed “ragged red fibers” which are indicators for defects in the respiratory chain of muscles [6].

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The defect sometimes causes changes in the behaviour of the affected animals such as aggressiveness, shying and panic in everyday situations. The first signs of BCSE can appear with an age of 6 months, but mostly the affected animals are not noticed prior first breeding. This eye anomaly is incurable [1].

In a previously performed whole genome scan using multipoint non-parametric linkage and haplotype analysis in a total of 159 German Brown cattle, we identified a genomic region harbouring a locus responsible for BCSE on BTA18 [7]. We mapped this BCSE-locus to a 6.83 cM interval (MARC-USDA linkage map) on the telomeric end of BTA18 between the microsatellites BMS2785 (72.01 cM) and BM6507 (78.84 cM) using linkage and haplotype analysis. The Zmean and LOD score peaked at the marker DIK5109 (77.60 cM) [5]. This BCSE region corresponds to a 7.77 Mb interval between 55.23 (BMS2785) and 63.0 Mb (BM6507). These marker positions were determined using BLAST analysis for Btau_4.0 (Bos taurus genome assembly 4.0).

We could identify mis-innervation syndromes in humans with similarities in pathology and clinical features to BCSE in cattle. Progressive external ophthalmoplegia (PEO), Duane retraction syndrome (DRS) and congenital fibrosis of the extraocular muscles (CFEOM) belong to this group of diseases in man. PEOs are characterized by slowly progressive bilateral immobility of the eyes accompanied by ptosis. The three candidate genes POLG [8], ANT1 [9] and C10orf2 [10] for PEO were ruled out as responsible for BCSE [1, 11]. CFEOM [12] and DRS [13, 14] belong to a group of congenital cranial nerve dysinnervation disorders (CCDD) affecting the eye, eye lid and/or facial movement [15]. The various forms of CFEOM [12] result from dysinnervation of oculomotor and/or trochlear nerve innervated ocular muscles.

Genes or loci causing CFEOM phenotypes include KIF21A (CFEOM1) on centromeric HSA12q12 [16, 17], ARIX (CFEOM2) on HSA11q.13.3-q13.4 [18], CFEOM3 on HSA16q24.2-q24.3 [19] and CFEOM3A on HSA12p11.2-q12 [20]. The bovine syntenic regions for these genes or loci are on BTA5, 9.7 Mb distally of the QTL for BCSE (KIF21A), on BTA15 at 51.34 Mb (ARIX) and on BTA18 from 11.5 to 14.0 Mb (CFEOM3). The loci for DRS were mapped to HSA8q13 (DURS1) [21-23]

and HSA2q31 (DURS2) [24, 25]. The orthologous bovine loci are on BTA14 between 30.2 and 30.7 Mb (DURS1) and BTA2 between 14.7 and 21.3 Mb (DURS2).

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Therefore, none of these loci or genes identified for CCDD in man are mapping within the QTL for BCSE.

Comparison of the gene order on the telomeric end of BTA18 (Btau_4.0) with the corresponding region on HSA19 (NCBI Build 36.2) showed two blocks of synteny (Figure 1). The gene order within the first block from LOC540740 to PRKCG is consistent with the human gene order. The second block between EPN1 (epsin 1) and TFPT (TCF3 fusion partner) (Btau_4.0) is inversed in comparison to the gene order of the human genome assembly 36.2. In our analysis we considered the interval from LOC540740 (54.98 Mb) to TFPT (63.54 Mb) which included the linked BCSE-region and its flanking regions on BTA18.

The aim of this study was to identify single nucleotide polymorphisms (SNPs) associated with BCSE within the previously determined BCSE region and within the coding sequence of possible candidate genes contained in this region. Candidate genes were chosen due to their expression profile and their proximity to the microsatellite DIK5109.

The first candidate gene, troponin T type 1 (TNNT1) is located about 200 kb proximal of DIK5109 at 62.50 Mb. The protein product of TNNT1 is a component of the thin filament of the sarcomere and has the function to prevent actin-myosin interaction in resting muscle. TNNT1 is highly expressed in skeletal muscles [26]. The second candidate gene, retinol dehydrogenase 13 (RDH13) is located in close vicinity of TNNT1 at 62.70 Mb. RDH13 belongs to the short-chain dehydrogenases/reductases (SDR) family and is mostly expressed in cranial nerve tissue and also in the retina, where it was detected in the inner segment of the photoreceptor cells [27]. Mutations causing strabismus have not yet been reported, but related genes such as RDH5 and RDH12 were shown to cause fundus albipunctatus and retinal dystrophy in human, which can be accompanied by strabismus [28, 29]. The third candidate gene, TCF3 fusion partner (TFPT), is ubiquitously expressed, mainly in brain, hemopoietic cell lines, and also in eye tissue.

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3.3 Material and Methods

3.3.1 Animals, phenotypic data and DNA/RNA extraction

For our analyses, we collected blood samples of 96 unrelated German Brown cows.

Of these animals, 48 were affected by BCSE and showed a third or fourth stage BCSE, where more than 50% of the eye was filled with sclera [30]. The other 48 German Brown cows were unaffected and more than 6 years old. Thus, these animals are very unlikely to develop the BCSE phenotype. Genomic DNA from EDTA blood samples was extracted using the QIAamp 96 Spin Blood Kit (Qiagen, Hilden, Germany).

For cDNA analysis, we took biopsies from retina, N. opticus and ocular muscles (M.

rectus lateralis and M. retractor bulbi) of three unaffected and three severely affected cows (stage 3 of BCSE) [30]. These samples were taken 15-30 minutes after the cows were slaughtered.

Tissue samples were conserved using RNA-later solution (Qiagen). The RNA was extracted from the ocular tissues using the Nucleospin RNA II-Kit (Macherey-Nagel, Düren, Germany) and transcribed into cDNA using SuperScript III Reverse Transcriptase (Invitrogen, Karlsruhe, Germany).

3.3.2 Gene structure, single nucleotide polymorphisms, PCR and DNA sequencing

Bioinformatic cDNA analysis: For cDNA analyses of the candidate genes, we searched the cattle expressed sequence tag (EST) archive for ESTs and the bovine genome

(http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=9913) for annotated genes by cross-species BLAST searches with the corresponding human reference mRNA sequences for TNNT1 (NM_003283), RDH13 (NM_138412) and TFPT (NM_013342). Table 1 gives an overview of the structure of these human genes and their orthologues in Bos taurus. In addition, we verified the sequence homology between the proteins of the three candidate genes in cattle, mouse and

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human using ClustalW alignment program (http://www.ebi.ac.uk/Tools/clustalw/index.html) (Figure 2).

We found a bovine EST (EE371552), isolated from muscle tissue with 89% identity to the human TNNT1 mRNA sequence, and the bovine mRNA of TNNT1 (NM_174474) with an identity of 90% to the human mRNA (NM_003283).

For RDH13 we found two overlapping bovine ESTs (DV925005 and DV828503) which cover 77% of the human mRNA sequence with an identity of 88% and the bovine mRNA of RDH13 (NM_001075345). The first EST (DV925005) was isolated from the skin of an embryo and the second (DV828503) from fetal pons.

We found three overlapping bovine ESTs (DV851209, CO881320 and CO873631), isolated from brain tissue, covering the whole human TFPT mRNA sequence with an identity of 86%. Furthermore, we identified the bovine TFPT gene employing a genomic BLAST analysis with the bovine mRNA sequence (NM_001075274).

We amplified the cDNA sequence corresponding to the open reading frames (ORF) of the three candidate genes. We used the ESTs and the annotated gene information for primer design with Primer3 software (http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi) (Table 2).

Genomic DNA sequence analysis for SNP detection: For these analyses we employed four BCSE-affected German Brown cows and four controls of the same breed. First, we designed exon flanking intronic primer pairs for the genomic amplification of all exons of RDH13 and the exons of TNNT1 and TFPT which harboured SNPs detected by cDNA analyses (Table 3). Furthermore, we designed primer pairs for four PCR-products of these candidate genes for SNP detection within intronic regions (Table 4). In order to cover the whole region of 8.56 Mb extending between LOC540740 and TFPT, we screened further 17 genes for DNA polymorphisms. A total of 21 amplicons was sequenced (Table 4). We used the DNA of eight German Brown cows (four affected and four controls) for SNP development in the three positional candidate genes and the 17 genes evenly distributed over the QTL region.

PCR and DNA sequencing: Here, we used 48 BCSE-affected German Brown cows and 48 unaffected cows of the same breed. PCR reactions were performed in a total volume of 30 µl using 2 µl (~ 20 ng/µl) genomic DNA, 3 µl 10x PCR buffer, 6 µl

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10xPCR Enhancer (PeqLab, Erlangen,Germany) 0.6 µl (10 µM) of each primer, 0.6 µl dNTPs (10mM each) and 0.2 µl (5 U/µl) Taq Polymerase (Roche, Mannheim, Germany). The reactions were performed in TProfessional thermocyclers (Biometra, Goettingen, Germany) and started with 5 min initial denaturation at 95°C, followed by 36 cycles at 95°C for 30 sec, optimum annealing temperature (Ta) at 58-60°C for 1 min, and extension at 72°C for 45 sec. The PCR was completed with a final cooling at 4°C for 10 min. After purification of the PCR products with MinElute 96 UF Plate (Qiagen), the amplicons were directly sequenced with the DYEnamic ET Terminator Cycle Sequencing kit (GE Healthcare, Freiburg, Germany) on a MegaBACE 1000 capillary sequencer (GE Healthcare). Sequence data was analyzed using the Sequencher 4.7 program (GeneCodes, Ann Arbor, MI, USA).

We analyzed a total of 41 PCR-products within 20 genes (Tables 2-4). We genotyped all 20 SNPs detected in the cDNA and genomic sequences of the three candidate genes and the nine SNPs detected within additional genes in the BCSE region for the complete sample of 48 BCSE affected German Brown cows and 48 unaffected cows of the same breed (Table 5).

3.3.3 Statistical analyses

A case-control analysis based on χ²-tests for genotypes, alleles and trend of the alleles was performed using the CASECONTROL procedure of SAS/Genetics [SAS, version 9.1.3 (Statistical Analysis System, Cary, NC, USA)]. The ALLELE procedure of SAS was used for estimation of allele frequencies and tests for Hardy-Weinberg equilibrium of genotype frequencies. Statistical calculation of pairwise linkage disequilibrium (LD) was performed and pictured using HAPLOVIEW 4.0 (http://www.broad.mit.edu/mpg/haploview/) [31]. We used the Tagger algorithm r²≥0.8 [32] to detect SNPs with strong LD among alleles. Subsequently, the association of haplotypes with BCSE was tested using the HAPLOTYPE procedure of SAS/Genetics.

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3.4 Results

3.4.1 Hardy-Weinberg equilibrium (HWE) and minor allele frequencies In total, we developed 29 SNPs within twelve genes. Of these 29 SNPs, 20 were located within the three candidate genes TNNT1, RDH13 and TFPT. The other nine SNPs were discovered in nine different genes located in the 8.56 Mb interval between LOC540740 (similar to inward rectifier potassium channel) and TFPT. The genotypic distributions of the 27 genotyped SNPs were in Hardy-Weinberg equilibrium. The SNPs within LOC540740 and exon 11 of TNNT1 were not in Hardy- Weinberg equilibrium (HWE). Thus, these SNPs were not considered in the subsequent association analyses. The results of the tests for HWE, the observed heterozygosity (HET), polymorphism information content (PIC) and minor allele frequencies for the developed SNPs are shown in Table 5.

3.4.2 Mutation analysis within the bovine TNNT1, RDH13 and TFPT gene

We revealed a total of ten exonic SNPs within the three candidate genes and a new splice variant of the TNNT1 gene (Table 5). Furthermore, we detected ten SNPs in the intronic sequences of these candidate genes (Figures 3 and 4).

TNNT1

Two exonic SNPs are located in the coding sequence of TNNT1 gene. One SNP was found in exon 11 and the second in exon 13. Both SNPs did not affect the amino acid sequence. We also identified a deletion of 33 base pairs in the cDNA sequence from eye muscle tissue of all six cows. These 33 base pairs conform to exon 4 of the published bovine mRNA (NM_174474) (Figure 4). In contrast, the cDNA isolated from retina, showed that all tested animals were heterozygous for this splice variant. In nerve tissue all three genotypes were found. In addition, we found one SNP within intron 10.

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RDH13

In the RDH13 gene, five exonic SNPs were detected. An A>C transversion (AM930555:c.425A>C) is located in the 5’UTR 14 bases upstream of the start codon.

A C>G SNP (AM930553:c.103C>G) is located at position 151 of bovine mRNA (NM_001075345) in exon 1. This SNP changes a CGG triplet to a GGG triplet and thus causes an amino acid exchange from arginine to glycine (p.Arg11Gly). This means a change from a charged alkaline amino acid to a nonpolar amino acid. The second SNP (AM930554:c.491G>C), which results in an amino acid exchange from glutamine to glutamate (p.Gln233Glu), was found at position 33 of bovine exon 7.

This G>C transversion changes a GAG triplet to a CAG triplet, which has the effect that a polar and uncharged amino acid is replaced by an acidic, unpolar and charged amino acid in the primary structure of the protein. In addition, we detected two synonymous SNPs in the coding sequence of exon 2 and 5. Within the introns of RDH13 we detected each one SNP in intron 1, 2 and 3 and two SNPs each in intron 5 and 6 (Table 5).

TFPT

In the coding sequence of the TFPT gene, we identified three exonic SNPs. Two of them are located at position 2 and 44 of exon 2. Both mutations affect the protein structure. The first exon 2 SNP (AM930551:c.337A>T) is an A>T transversion which causes an amino acid exchange from threonine to serine (p.Thr9Ser). But both amino acids are polar, uncharged and differ in only one ─CH3 side chain. The second SNP (AM930551:c.379G>T) in exon 2 alters the protein structure due to a G>T transversion which changes a GGC triplet to a TGC triplet (p.Gly23Cys). This means that the unpolar amino acid glycine is exchanged with the polar sulfur containing amino acid cysteine. The third exonic SNP found in the ORF of TFPT is a synonymous mutation. This G>A SNP (AM930552:c.176G>A) at position 72 in exon 5 changes a CTG to a CTA triplet, which has no effect on the amino acid sequence of TFPT. In addition, we identified each one SNP in intron 2 and 3 of TFPT.

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3.4.3 Association analysis

We detected four SNPs significantly associated with BCSE. These were located in the DHDH, CPT1C, TNNT1 and NALP7 genes (Table 5 and 6). An exonic A>G transition (AM930555:c.359A>G) within TNNT1 reached significant results in allele and trend test statistics (Table 6).

The SNPs within the exons and the exon flanking intronic sequences of RDH13 showed no significant results from the χ²-tests for distribution of genotypes between cases and controls. The χ²-test statistics for allelic distributions between cases and controls ranged from 0.003 to 3.20 and their error probabilities from 0.07 to 0.97 for the RDH13-SNPs. Four exon-SNPs failed clearly the threshold of significance. Only the C>A SNP (AM930553:c.703C>A) in exon 5 with an allelic χ²-value of 3.20 was close to the threshold of 0.05 (Table 6).

The exonic SNPs of TFPT were not genotyped for the complete sample due to their low minor allele frequency (Table 5) and the other intronic SNPs were not associated with BCSE (Table 6).

3.4.4 Linkage disequilibrium (LD) and haplotype association

The r²-values indicated strong linkage disequilibrium (LD) for the SNPs between intron 1 and intron 2 of the RDH13 gene. By tagging with threshold r²≥0.8, we detected five SNPs in RDH13 which were representative for the total of twelve RDH13 SNPs. Therefore, only these five SNPs of RDH13 were used in the haplotype association analysis. The SNPs within the other genes were not in LD (Figure 5).

We tested the association of haplotypes with BCSE, including three to eight SNPs and permutating the number of SNPs which were in Hardy-Weinberg equilibrium. The marker-trait association, including five SNPs located in the genes CPT1C (AM930539:g.569A>G), SYT5 (AM930544:g.71G>A), RDH13 (AM930553:c.703C>A and AM930547:g.194C>T), NALP7 (AM930543:g.103T>G) was significant (χ²=54.11, p<0.0001). In total, there were 8 different haplotypes of these markers which had a frequency of at least 1% (Table 7). Three individual haplotypes were significantly associated with the affection status and occurred with a frequency of more than 5%

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in our sample. The A-G-C-C-G haplotype occurred in our sample of affected cows with a frequency of 31.7% and with 9.4% in the controls. The A-G-A-T-T haplotype occurred with a frequency of 17.1% in the sample of unaffected cows and with 6.1%

in affected animals. The third associated haplotype (G-G-C-C-T) was found with a frequency of 17.0% in our sample of unaffected cows and with a frequency of 7.0% in the affected animals (Table 7). The significantly associated haplotypes spanned the region from CPT1C (56.05 Mb) to NALP7 (62.87 Mb) on the telomeric end of BTA18.

Further two haplotypes adjacent to the proximal and distal region of this aforementioned associated region were tested for association with BCSE. The first haplotype proximally to CPT1C consisted of the SNPs (AM922316:g.141C>G, AM930537:g.351A>C, AM930538:g.493T>C) and the second haplotype distally to NALP7 included the SNPs (AM930542:g.365G>A, AM930541:g.120T>C, AM930541:g.342G>A). Both adjacent haplotypes did not show significant results in marker-trait association tests with BCSE (χ²=11.7, p =0.07 and χ²=7.3, p=0.12).

3.5 Discussion

We developed a total of 29 intragenic SNPs within an 8.56 Mb region on BTA18 extending from LOC540740 to TFPT. In a previously performed whole genome scan, this region was found to be linked with BCSE in German Brown dairy cattle [7]. Within the genes CPT1C and NALP7 two SNPs were significantly associated with BCSE in association tests for single markers. Within DHDH, TTYH1 and SYT5, three further SNPs reached values close to the significance threshold of p=0.05.

Most of the 29 SNPs were detected in the sequences of the potential candidate genes RDH13, TFPT and TNNT1. We identified four missense mutations within the coding sequence of these three genes and also detected a new splice variant of TNNT1. Since none of the SNPs within the genes RDH13, TFPT and TNNT were significantly associated with BCSE in association tests for single markers, these genes are unlikely to be causal for this eye defect. However, the detected polymorphisms may be of importance in studies for other bovine diseases. Especially the SNPs within RDH13 could be involved in the genetic pathology of retinal

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dystrophy or related diseases like the defects reported to be associated to other members of the short-chain dehydrogenases/reductases (SDR) family [27, 28].

In cattle, genes influencing the development of strabismus are not yet known.

Therefore, neuromuscular eye-disorders in man, with already identified causal genes and even developmental and biological characteristics, may be used as candidates for BCSE. However, all potential candidate genes characterized as causal for these syndromes (PEO, CFEOM and DRS) in man [11-25] could be ruled out for bovine BCSE because we could not find linkage or these candidate genes did not map in the BCSE-linked region on BTA18.

We employed haplotype analysis to further refine the BCSE region on BTA18. In order to find the most likely associated haplotype, we permutated the number of SNPs for the different haplotypes within this region and were then able to find a significantly associated haplotype. This haplotype included SNPs from the genes CPT1C, SYT5, RDH13, and NALP7. Presence of the haplotype A-G-C-C-G composed of these five SNPs indicated a high probability of an animal to be affected by BCSE later in life, whereas the haplotypes A-G-A-T-T and G-G-C-C-T were related with low risk to BCSE. Because the surrounding SNPs did not contribute to the significance of the haplotype association, confirmation has been obtained that this linked BCSE region could be delimited using haplotype analysis. Robustness of the haplotype association was furthermore evident when the surrounding haplotypes were extended with one or three adjacent SNPs from the associated haplotype region. In these cases, the extended haplotypes reached higher χ²-test statistics and lower error probabilities as more SNPs of the associated region were included. This result was according to our expectation for this region. In conclusion, the haplotype association refined the BCSE-region to a 6.82 Mb interval.

In order to detect the gene responsible for bovine BCSE, further SNPs have to be developed within this BCSE region spanning from 56.05 Mb to 62.87 Mb on BTA18.

Haplotype analysis may then be a valuable tool to determine the most likely BCSE causing gene.

Particularly, SNPs within potential candidate genes like CPT1C will be considered.

CPT1C is located at 54.89 Mb on HSA19 and specifically expressed within endoplasmatic reticulum (ER) in neurons of brain [33]. Expression was also detected

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in the retinal pigment epithelium [34]. The function of this gene is not yet clearly defined. CPT1C is believed to modulatethe palmitoyl-CoA pool associated with the ER, thus regulating the synthesis of ceramide and sphingolipids. Ceramide and sphingolipids are important for signal transduction, modification of neuronal membranes,and brain plasticity [35-37]. Since one of the BCSE-associated SNPs is located within intron 15 of bovine CPT1C at 56.05 Mb on BTA18, this gene may be a candidate for BCSE.

3.6 Acknowledgements

This study was supported by a grant of the German Research Council, DFG, Bonn, Germany (DI 333/7-3). The authors thank all breeders and veterinarians for their readiness to support collection of blood samples of affected animals and controls. We particularly thank Dr. F. Merz and the other veterinarians of abattoir in Buchloe (Germany) for their support. We also thank Heike Klippert-Hasberg and Stefan Neander for expert technical assistance.

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